Genome-wide end-sequenced BAC resources for the NOD/MrkTac() and NOD/ShiLtJ() mouse genomes.

Abstract

Non-obese diabetic (NOD) mice spontaneously develop type 1 diabetes (T1D) due to the progressive loss of insulin-secreting beta-cells by an autoimmune driven process. NOD mice represent a valuable tool for studying the genetics of T1D and for evaluating therapeutic interventions. Here we describe the development and characterization by end-sequencing of bacterial artificial chromosome (BAC) libraries derived from NOD/MrkTac (DIL NOD) and NOD/ShiLtJ (CHORI-29), two commonly used NOD substrains. The DIL NOD library is composed of 196,032 BACs and the CHORI-29 library is composed of 110,976 BACs. The average depth of genome coverage of the DIL NOD library, estimated from mapping the BAC end-sequences to the reference mouse genome sequence, was 7.1-fold across the autosomes and 6.6-fold across the X chromosome. Clones from this library have an average insert size of 150 kb and map to over 95.6% of the reference mouse genome assembly (NCBIm37), covering 98.8% of Ensembl mouse genes. By the same metric, the CHORI-29 library has an average depth over the autosomes of 5.0-fold and 2.8-fold coverage of the X chromosome, the reduced X chromosome coverage being due to the use of a male donor for this library. Clones from this library have an average insert size of 205 kb and map to 93.9% of the reference mouse genome assembly, covering 95.7% of Ensembl genes. We have identified and validated 191,841 single nucleotide polymorphisms (SNPs) for DIL NOD and 114,380 SNPs for CHORI-29. In total we generated 229,736,133 bp of sequence for the DIL NOD and 121,963,211 bp for the CHORI-29. These BAC libraries represent a powerful resource for functional studies, such as gene targeting in NOD embryonic stem (ES) cell lines, and for sequencing and mapping experiments.

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Genome-wide end-sequenced BAC resources for the NOD/
MrkTac☆ ☆ and NOD/ShiLtJ☆☆☆☆ mouse genomes
Charles A. Stewarda,⁎, Sean Humphraya, Bob Plumba, Matthew C. Jonesa, Michael A.
Quaila, Stephen Ricea, Tony Coxa, Rob Daviesa, James Bonfielda, Thomas M. Keanea,
Michael Nefedovc, Pieter J. de Jongc, Paul Lyonsd, Linda Wickerd, John Toddd, Yoshihide
Hayashizakie, Omid Gulbanf, Jayne Danskaf, Jen Harrowa, Tim Hubbarda, Jane Rogersb, and
David J. Adamsa
aThe Wellcome Trust Sanger Institute, Hinxton, UK.
bThe Genome Analysis Centre, Norwich Research Park, Norwich, UK.
cChildren's Hospital Oakland Research Institute, Oakland, CA, USA.
dCambridge Institute for Medical Research, Cambridge, UK.
eRIKEN, Yokohama Institute, Japan.
fHospital for Sick Children Research Institute and University of Toronto, Toronto, Canada.
Abstract
Non-obese diabetic (NOD) mice spontaneously develop type 1 diabetes (T1D) due to the progressive
loss of insulin-secreting β-cells by an autoimmune driven process. NOD mice represent a valuable
tool for studying the genetics of T1D and for evaluating therapeutic interventions. Here we describe
the development and characterization by end-sequencing of bacterial artificial chromosome (BAC)
libraries derived from NOD/MrkTac (DIL NOD) and NOD/ShiLtJ (CHORI-29), two commonly used
NOD substrains. The DIL NOD library is composed of 196,032 BACs and the CHORI-29 library is
composed of 110,976 BACs. The average depth of genome coverage of the DIL NOD library,
estimated from mapping the BAC end-sequences to the reference mouse genome sequence, was 7.1-
fold across the autosomes and 6.6-fold across the X chromosome. Clones from this library have an
average insert size of 150 kb and map to over 95.6% of the reference mouse genome assembly
(NCBIm37), covering 98.8% of Ensembl mouse genes. By the same metric, the CHORI-29 library
has an average depth over the autosomes of 5.0-fold and 2.8-fold coverage of the X chromosome,
the reduced X chromosome coverage being due to the use of a male donor for this library. Clones
from this library have an average insert size of 205 kb and map to 93.9% of the reference mouse
genome assembly, covering 95.7% of Ensembl genes. We have identified and validated 191,841
single nucleotide polymorphisms (SNPs) for DIL NOD and 114,380 SNPs for CHORI-29. In total
© 2010 Elsevier Inc.
This document may be redistributed and reused, subject to certain conditions.
⁎Corresponding author. Wellcome Trust Sanger Institute Wellcome Trust Genome Campus Hinxton, Cambridge, CB10 1HH, UK. Fax:
+44 0 1223 494919. cas@sanger.ac.uk.
This document was posted here by permission of the publisher. At the time of deposit, it included all changes made during peer review,
copyediting, and publishing. The U.S. National Library of Medicine is responsible for all links within the document and for incorporating
any publisher-supplied amendments or retractions issued subsequently. The published journal article, guaranteed to be such by Elsevier,
is available for free, on ScienceDirect.
☆ Sequence data from this library have been deposited with the DDBJ/EMBL/GenBank Data libraries under Accession Numbers
FR000001–FR332535.
☆☆ Sequence data from this library have been deposited with the DDBJ/EMBL/GenBank Data libraries under Accession Numbers
FR332536–FR502694.
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we generated 229,736,133 bp of sequence for the DIL NOD and 121,963,211 bp for the CHORI-29.
These BAC libraries represent a powerful resource for functional studies, such as gene targeting in
NOD embryonic stem (ES) cell lines, and for sequencing and mapping experiments.
Keywords
Bacterial artificial chromosome; NOD/MrkTac; NOD/ShiLtJ; Mouse genome; Non-obese diabetic
(NOD); Type 1 diabetes; T1D; Insulin-dependent diabetes; IDD
Introduction
Type 1 diabetes (T1D) or insulin-dependent diabetes (IDD) is a polygenic disorder,
characterized by hyperglycaemia that results from the autoimmune T cell-mediated destruction
of the insulin-producing β-cells of the islets of Langerhans of the pancreas [1–3]. T1D is
triggered by different environmental and genetic factors and has variable penetrance,
suggesting that susceptibility to this syndrome is inherited and polygenic [1–6].
The non-obese diabetic (NOD) mouse is an experimental model for human T1D, developed in
Japan by Makino et al. [7]. NOD mice spontaneously develop T1D following an autoimmune
mediated process that progressively destroys their insulin-secreting β-cells [2,3]. T1D is
typically associated with allelic variants of the Human Leukocyte Antigen (HLA) class II
immune response genes within the Major Histocompatability Complex (MHC) [3,8]. Genetic
analysis of the NOD mouse has further established that the inheritance of diabetes in these
mice is controlled polygenically by at least 27 disease-associated loci, distributed over at least
14 different chromosomes [9]. These loci have been designated Idd loci, for insulin-dependent
diabetes [9,10].
Understanding how these loci contribute to the development of T1D in the NOD mouse should
inform us of the underlying mechanisms of T1D development in humans. It is important,
however, to analyse Idd loci in the context of the genome in which they reside so that the effect
of the background in which the phenotype is observed, and the role of epistatic genetic
interactions can be assessed. Bacterial artificial chromosomes (BACs) represent a useful
resource for sequencing, mapping and functional studies [11]. Here we describe the
development and end-sequencing of two BAC libraries for the NOD substrains NOD/MrkTac
(DIL NOD) and NOD/ShiLtJ (CHORI-29). While NOD/MrkTac and NOD/ShiLtJ mice are
derived from the same founding stock of NOD mice developed by intercrossing Jcl:ICR
(Institute for Cancer Research) mice for more than 20 generations [7] they have been
maintained as isolated colonies for many generations, and as such are likely to have diverged
significantly. Indeed these NOD substrains show subtle differences in the timing and
presentation of diabetes, and also in their plasma glucose levels. The availability of BAC
libraries for both of these NOD substrains will allow us to study the differences between them
and to gain a better understanding of the pathogenesis of T1D. In addition, with the recent
advent of embryonic stem (ES) cells derived from NOD mice [12,13] these BAC libraries will
form the foundation for targeted manipulation of the NOD mouse genome.
Results
End-sequencing
All clones from the DIL NOD and CHORI-29 BAC libraries were end-sequenced and the
sequence read data have been submitted to EMBL. These data are also available from the
Ensembl trace repository (http://trace.ensembl.org/) and the NCBI Trace Archive
(http://www.ncbi.nlm.nih.gov/Traces/trace.cgi). 332,535 DIL NOD BAC clone end-sequences
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successfully passed post-sequencing quality processing from a total of 196,032 BACs,
generating 229,736,133 bp of sequence. Of these passed reads, 318,065 (95.6%) were aligned
to the C57BL/6J reference genome (NCBIm37), 170,029 (53.5%) of which were aligned to a
single definitive location (Table 1A). Similarly for the CHORI-29 library, 170,159 BAC clone
end-sequences passed post-sequencing quality processing from 110,976 BACs, generating
121,963,211 bp of sequence. Of these passed reads, 159,574 (93.8%) were aligned successfully
to the reference C57BL/6J genome with 80,710 (50.6%) reads aligned to a single definitive
location on NCBIm37 (Table 1B). The majority of the reads that did not map contained
repetitive sequences or were of low quality. Both sets of data can be downloaded from the
Sanger FTP site (ftp://ftp.sanger.ac.uk/pub/NODmouse/NOD_BACend_alignments).
Mapping was performed using SSAHA2 with default parameters [14]. Using read-pair
information we could place 41,468 DIL NOD clones and 18,257 CHORI-29 clones
unambiguously on the genome since both read-pairs matched uniquely. However, it was also
possible to establish the position of certain clones for which only one end mapped uniquely
where the other end of the clone mapped to the genome within 3 standard deviations of the
mean insert length of clones from the library and on the opposite sequence strand. This allowed
us to place a further 83,796 DIL NOD clones and 43,905 CHORI-29 clones on the genome,
resulting in a total of 125,266 uniquely placed DIL NOD clones and 62,162 uniquely placed
CHORI-29 clones. The different success rates in the unique positioning of the BAC clones
from these libraries to the genome was largely due to differences in the quality of end-sequence
data produced from both libraries. CHORI-29 clones have larger genomic inserts and as a
consequence were harder to prep and sequence compared to clones from the DIL NOD BAC
library. Using the mapping data for both libraries it was possible to estimate the average insert
size for the DIL NOD library to be 149,809 bp and 205,413 bp for the CHORI-29 library (Fig.
1A), which correlated with the experimentally derived figures (Fig. 1B).
Physical genome coverage and coverage of Ensembl genes
The average depth of genome coverage of the end-sequenced DIL NOD library was calculated
to be 7.1-fold across the autosomes and 6.6-fold across the X chromosome (Table 2A). The
end-sequenced CHORI-29 library has an average depth of 5.0-fold and 2.8-fold across the
autosomes and the X chromosome, respectively (Table 2B). The total number of Ensembl
[15] predicted genes that are fully covered by a BAC clone from the DIL NOD library is 31,093
(98.8%) and 30,103 (95.7%) for the CHORI-29 library, based upon Ensembl mouse release
55 (NCBIm37). The total number of Ensembl genes partially covered by DIL NOD and
CHORI-29 BACs is 349 (1.1%), and 1,351 (4.3%) respectively, 200 (0.6%) of which are partial
in both libraries. The total number of Ensembl genes contained completely in DIL NOD and
CHORI-29 BAC gaps is 30 (0.1%) and 18 (0.06%) respectively. There are 5 (0.02%) genes
that are present on the reference genome but absent completely from both NOD libraries. This
is likely because it was not possible to place one or both BAC end-reads of a pair due to
reference genome gaps adjacent to these Ensembl genes. These data are available on the Sanger
FTP site (ftp://ftp.sanger.ac.uk/pub/NODmouse/NOD_Ensembl_gene_coverage).
To make the data accessible to the wider community, we have generated a Distributed
Annotation System (DAS) [16] source to display both the DIL NOD clones
(http://www.ebi.ac.uk/das-srv/genomicdas/das/nod_clones_m37) and the CHORI-29 clones
(http://www.ebi.ac.uk/das-srv/genomicdas/das/chori29_clones_m37) so that they can be
visualized in DAS source compliant browsers. For example, in the Ensembl genome browser
(http://www.ensembl.org/Mus_musculus/Info/Index) the alignments of both NOD BAC
libraries can be accessed through the DAS sources menu and viewed against the reference
C57BL/6J genome. DIL NOD clones are displayed as red and black lines depending on the
orientation of the insert in the vector, while CHORI-29 clones are displayed as green and blue
lines. The BAC end-sequences can also be viewed as traces in the main “Region in Detail”
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window of the Ensembl genome browser. The method used to display BAC ends in Ensembl
shows only those that have corresponding ends that are considered to be within 3 standard
deviations of the mean insert size of the library. End-reads provide a link to the Ensembl trace
repository (http://trace.ensembl.org), where the end-read sequences for all quality clipped reads
have been deposited (Fig. 2). FASTA files of these quality clipped reads have also been
generated and deposited on the Sanger FTP site
(ftp://ftp.sanger.ac.uk/pub/NODmouse/NOD_BACend_fasta_sequences).
Analysis of nucleotide variation
We used SSAHA-SNP2 [15] to call single nucleotide polymorphisms (SNPs) and deletion
insertion polymorphisms (DIPs) from both the DIL NOD and CHORI-29 end-sequence reads
by comparing them to the NCBIm37 C57BL/6J assembly. We called 191,841 SNPs and 15,824
DIPs for DIL NOD and 114,380 SNPs and 4,942 DIPs for CHORI-29. These data are available
on the Sanger FTP site (ftp://ftp.sanger.ac.uk/pub/NODmouse/NOD_variation_data). The
following criteria were used: the identity had to be greater than or equal to 92% match length,
greater than or equal to 80% of the read length and a match score or match length greater than
250 bp. These SNPs have been validated against Illumina whole genome shotgun data of the
NOD/ShiLtJ genome (http://www.sanger.ac.uk/modelorgs/mousegenomes/) and submitted to
dbSNP. However, it is important to note that the DIPs are candidate nucleotide variants and
follow-up genotyping is warranted.
Discussion
The genome-wide DIL NOD and CHORI-29 mouse BAC end-sequenced libraries provide a
unique way of studying T1D in mouse. The two libraries were aligned against the C57BL/6J
mouse genome and are displayed on the Ensembl genome browser, which almost eliminates
the need to perform filter hybridizations to isolate clones of interest, except for “non-reference”
regions of the genome that are novel in NOD mouse. The distribution of mapped sequence
reads is relatively even across the genome, with the exception of the Y chromosome, which is
not represented in the DIL NOD mouse library. To date, high-quality BAC libraries exist for
several mouse strains, including C57BL/6J, MSM/Ms [17,18], C3H/HeJ, BALB/c, A/J,
SPRET/Ei, AKR/J, CAST/Ei (http://bacpac.chori.org/home.htm) and 129S7 [11], with BAC
end-sequences existing for C57BL/6J, MSM/Ms and 129S7. Such libraries have been shown
to be an invaluable resource for assembling genomes and for in vivo functional studies, such
as BAC rescue [19–21].
The high-density and end-sequence quality of these BAC libraries make them useful tools for
examining large-scale structural differences between the two substrains of NOD and other
mouse strains and will greatly facilitate high-throughput targeted manipulation of the NOD
mouse genome. With the recent advent of NOD ES lines these BAC libraries will be a critical
resource for targeting vector construction [12,13] where isogenic DNA has been shown to be
critically important in obtaining high targeting frequencies [22]. Due to the high coverage of
the C57BL/6J reference genome by the NOD BAC ends, regions with poor coverage of the
reference genome may represent structural variants in the NOD mouse genome when compared
to the reference genome. Importantly we are currently performing targeted sequencing of
regions of the NOD mouse genome relevant to T1D, which is crucial for understanding the
role genetic susceptibility plays in the pathogenesis of T1D
(http://www.sanger.ac.uk/Projects/M_musculus-NOD/). We are also in the process of using
the Illumina platform [23] to sequence the entire NOD/ShiLtJ genome, which will greatly
improve the utility of these BAC resources, and should help to position unaligned BACs to
novel ‘non-reference’ regions of the NOD genome
(http://www.sanger.ac.uk/modelorgs/mousegenomes/).
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Materials and methods
Construction of the BAC libraries
The DIL NOD BAC library was constructed from NOD/MrkTac female liver genomic DNA
at the Genomic Sciences Center, RIKEN in collaboration with the Diabetes and Inflammation
Laboratory at Cambridge University. EcoRI was used to partially digest whole genomic DNA
and the resulting fragments were cloned in pBACe3.6 as described previously [18]. These
clones are available from Dr Jayne Danska jayne.danska@sickkids.on.ca. The CHORI-29
library was constructed in a similar manner at the Children's Hospital, Oakland, California,
USA. NOD/ShiLtJ male kidney genomic DNA, obtained from the Jackson Laboratory, was
cloned in pTARBAC2.1 and these clones are available from http://bacpac.chori.org/. The
average insert size was experimentally verified using clamped hexagonal electric field (CHEF)
electrophoresis, a system similar to pulse field gel electrophoresis (PFGE) [24]. The marker
(M) (Fig. 1B) was a MidRange II PFG Marker – N3552S from New England BioLabs. For
storage purposes, the DIL NOD library was arrayed into 527 384-well plates and the CHORI-29
library was replicated into 672 384-well plates.
End-sequence profiling of the BAC resource
In total 378,896 reads were attempted for the DIL NOD library using T7 and SP6, M13-21 and
pUCR, and 3HPPSK and 3HPpur primers on the vector and big dye terminator chemistry.
207,321 reads were attempted for the CHORI-29 library using the T7 and SP6 primers on the
vector, and big dye terminator chemistry. Sequence-reads were subjected to processing using
Automated Sequence Preprocessing (ASP) [25]. The number of insertless clones was
determined to be 3% for DIL NOD and 1% for CHORI-29. Average read-lengths were
determined to be 694.08 bp in length for DIL NOD and 718.77 bp in length for CHORI-29.
End-sequence mapping of BAC clones
End-read data were mapped using SSAHA2 with the mapping criteria that more than 100 bp
should map with greater than 95% identity to the NCBIm37 assembly. Clone-ends were
iteratively aligned against the genome and after each round the average size of the clones was
calculated as well as the standard deviation. Clone-ends that were plus or minus three standard
deviations away from the mean were rejected. Clones with only one end aligned, ends that
were orientated in the same direction, or ends that lie at unrealistic distances were rejected.
Conflict of interest statement
The authors declare that they have no conflict of interest.
Acknowledgments
This research was conducted as part of the Immune Tolerance Network, which is jointly funded by the National Institute
of Allergy and Infectious Diseases, the National Institute of Diabetes, Digestive, and Kidney Disorders (both part of
the National Institutes of Health) and the Juvenile Diabetes Research Foundation. The project was also supported by
the Medical Research Council, UK and the Wellcome Trust Sanger Institute. D. J. Adams is supported by Cancer
Research, UK and the Wellcome Trust.
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Table 1A
BAC clones in library196,032
Attempted BAC clone end-reads378,896
BAC clone end-reads sequenced successfully 332,535
Passed BAC clones sequenced on both strands 150,878
BAC clone end-reads with an alignment to the genome 318,065
Aligned BAC clone end-reads with a unique match170,029
Aligned BAC clone end-reads with multiple matches 148,036
BAC clones positioned using unique end-read pairs41,468
BAC clones positioned uniquely125,266
Sequencing and alignment summary for the DIL NOD BAC library.
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Table 1B
BAC clones in library110,976
Attempted BAC clone end-reads 207,321
BAC clone end-reads sequenced successfully170,159
Passed BAC clones sequenced on both strands75,046
BAC clone end-reads with an alignment to the genome159,574
Aligned BAC clone end-reads with a unique match80,710
Aligned BAC clone end-reads with multiple matches 78,864
BAC clones positioned using unique end-read pairs18,257
BAC clones positioned uniquely62,162
Sequencing and alignment summary for the CHORI-29 BAC library.
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Table 2A
Chr. nameTotal chr.
length bp
Non-redundant clone-length bpClone-length total bpPassed
aligned
sequence
bp
Clone depth % total coverage
1 197,195,432189,902,3831,385,443,53313,593,2087.0396.3
2 181,748,087177,813,7741,348,761,74613,129,8017.4297.84
3159,599,783155,666,9061,151,145,10811,396,9117.2197.54
4155,630,120 150,659,3651,134,909,77010,944,7277.2996.81
5152,537,259146,223,1451,009,782,4489,773,9156.6295.86
6149,517,037145,684,3061,049,822,25710,281,0877.0297.44
7152,524,553140,866,4141,007,473,8699,637,7326.6192.36
8131,738,871123,985,369907,535,3298,826,0366.8994.11
9124,076,172120,302,728916,247,0388,800,5007.3896.96
10 129,993,255125,971,520897,618,5208,764,9816.9196.91
11121,843,856118,333,935924,494,0658,842,9557.59 97.12
12121,257,530117,098,461 854,759,2238,258,2267.0596.57
13120,284,312116,142,787888,982,4168,609,8167.3996.56
14125,194,864119,290,632846,844,9748,247,8116.7695.28
15103,494,974100,431,693 735,484,6687,116,9227.1197.04
16 98,319,15094,516,190692,930,1726,811,5647.0596.13
1795,272,65191,304,604690,760,3366,680,8007.2595.84
1890,772,031 86,713,096655,698,2036,460,4837.2295.53
1961,342,43057,670,646421,571,2364,064,957 6.8794.01
X166,650,296160,833,1081,095,695,03110,991,8376.5796.51
Y15,902,55500000
All data in Table 1 and Table 2 are derived from the NCBIm37 mouse assembly. The DIL NOD library covers over 95.6% of the mouse genome at
an average depth of 7.1-fold across the autosomes and 6.6-fold across the X chromosome. Column 2 is the C57BL/6J chromosome length. The non-
redundant clone-length field (column 3) is the non-redundant BAC clone sequence estimated for that chromosome, using both read-pairs. This may
count each base several times, the number of times being the number of BACs overlapping that position. The clone-length total field (column 4) is
the total sequence estimated for all the BACs for that chromosome, using both read-pairs. The passed aligned sequence column (5) is the total number
of sequenced bases per chromosome that have successfully passed quality control and been mapped. The clone depth field (column 6) is the clone-
length total (column 4) divided by the total chromosome length (column 2). The % total coverage (column 7) is the non-redundant clone-length total
(counting each base covered only once) as a percentage of the total chromosome length.
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Table 2B
Chr. Name Total chr.
length bp
Non-redundant clone-length bp Clone-length total bp Passed
aligned
sequence
bp
Clone depth% total coverage
1197,195,432 188,433,0011,046,993,130 7,724,4845.31 95.56
2 181,748,087175,429,538942,386,7906,916,0615.1896.52
3159,599,783154,711,914875,284,125 6,490,6615.4896.94
4155,630,120148,498,769 827,737,3536,127,9055.32 95.42
5152,537,259144,530,268733,517,2185,404,820 4.8194.75
6 149,517,037143,599,805 775,532,2995,768,1045.19 96.04
7 152,524,553135,008,655644,735,047 4,788,9964.23 88.52
8131,738,871121,214,563606,581,6644,522,1094.60 92.01
9124,076,172118,654,230598,157,8624,362,9924.8295.63
10129,993,255124,749,578 675,601,3604,987,6135.20 95.97
11121,843,856115,930,403605,273,3454,474,9624.9795.15
12121,257,530115,274,597604,649,3544,482,4874.9895.07
13120,284,312114,926,992609,116,2134,517,9885.06 95.55
14125,194,864116,442,268607,970,6234,531,8844.8593.01
15103,494,97499,239,967543,851,1523,998,7635.2595.89
16 98,319,150 92,840,807 534,474,1343,928,8895.4494.43
1795,272,65189,677,039468,395,8353,456,1444.9294.13
1890,772,03185,715,767456,115,712 3,373,9745.02 94.43
1961,342,43056,286,798291,811,6982,148,1574.7591.76
X166,650,296148,955,095469,399,6603,543,8692.8289.38
Y15,902,5552,427,2239,762,86680,060 0.6215.26
The CHORI-29 library covers 93.9% of the mouse genome at an average depth of 5.0-fold across the autosomes and 2.8-fold across the X chromosome.
For further information regarding the data, please see legend for Table 2A.
Published as: Genomics. 2010 February ; 95(2): 105–110.